Cancer is the second most common cause of death in the U.S. and accounts for one of every eight deaths worldwide. During 2010, the American Cancer Society estimated approximately 1,529,560 new cancer cases would be diagnosed in the U.S. alone, and an estimated 569,490 Americans would die from cancer. In 2008, an estimated 12.4 million new cancer cases were diagnosed, and 7.6 million people died from cancer worldwide. Although medical advances have improved cancer survival rates, there is a continuing need for new and more effective treatment.
Cancer is characterized by uncontrolled cell reproduction. Antimitotic agents and antimicrotubule agents have been explored as targets for cancer therapy because of their important role in the cell division cycle. The cell division cycle, which regulates the transition from quiescence to cell proliferation comprises four phases: G1, S phase (DNA synthesis), G2, and M phase (mitosis). Non-dividing cells rest in quiescent phase, G0. Inhibition of the mitotic machinery results in a diverse array of outcomes, primarily leading to cell death or arrest.
As the effect of antimitotic agents is not limited to cancer cells alone, the dose-limiting toxicities of these drugs in a clinical setting frequently manifest in rapidly dividing tissue and in the case of antimicrotubule agents are often accompanied by severe peripheral neuropathy in the case of antimicrotubule agents. Therefore, the narrow therapeutic index of antimitotic agents necessitates an understanding of the mechanism of action of these drugs to maximize the chances of rational development of these therapies.
Traditional antimitotic agents include those that directly interfere with microtubule dynamics, essential for mitotic spindle assembly and the subsequent alignment and segregation of DNA to daughter cells. Antimicrotubule agents, such as Taxanes are currently being used in clinical setting. For example, paclitaxel and docetaxel have a similar spectrum of clinical activity including ovarian, lung, breast, bladder, and prostate cancers.
Taxanes stabilize microtubules by altering the kinetics of microtubule depolymerization. In mammalian cells grown in culture, high concentrations of paclitaxel cause the stabilization of aggregated microtubules (Schiff and Horwitz (1980) Proc Natl Acad Sci USA 77:1561-1565). At lower concentrations that resemble exposures achieved in clinical settings, the primary effect of paclitaxel is to stabilize microtubules, and thereby dampen the dynamic instability of microtubules that is a requisite for efficient spindle assembly. As a result of this dampening, microtubules are unable to grow and shrink rapidly, and their ability to bind to condensed chromosomes during mitosis is compromised. Efficient chromosome alignment is thus affected, and this failure of chromosome alignment leads to mitotic delays mediated via the spindle assembly checkpoint.
The spindle assembly checkpoint ensures that chromosomes are properly aligned to the metaphase plate prior to the anaphase initiation where sister chromatids segregate to opposite poles. Interestingly, at low concentrations of paclitaxel, inefficient chromosome alignment has been shown to occur without prolonged mitotic arrest, and the effect of paclitaxel is thus not dependent on its ability to induce mitotic arrest or delays (Chen and Horwitz (2002) Cancer Res 62:1935-1938); Kelling et al. (2003) Cancer Res 63:2794-2801).
For paclitaxel as well as its analog docetaxel, in vitro studies have demonstrated the presence of abnormal DNA contents and cell death even at concentrations where prolonged mitotic arrest does not occur (Chen and Horwitz (2002) Cancer Res 62:1935-1938; Hernandez-Vargas et al. (2007) Cell Cycle 6:780-783; Hernandez-Vargas et al. (2007) Cell Cycle 6:2662-2668. Consistent with this finding, preclinical studies in xenograft models have failed to demonstrate a clear relationship between the degree of mitotic arrest and tumor growth inhibition (Gan et al. (1998) Cancer Chemother Pharmacol 42:177-182; Milross et al. (1996) J Natl Cancer Inst 88:1308-1314; Schimming et al. (1999) Cancer Chemother Pharmacol 43:165-172), and similar findings have been reported in a clinical setting (Symmans et al. (2000) Clin Cancer Res 6:4610-4617).
It has been well established that antimitotic compounds compromise the ability of cells to execute a successful division. Cells will either fail to divide with a prolonged mitotic arrest that leads directly to cell death, or they divide abnormally, with an unequal distribution of DNA (Gascoigne and Taylor (2008) Cancer Cell 14:111-122; Rieder and Maiato (2004) Dev Cell 7:637-651; Weaver and Cleveland (2005) Cancer Cell 8:7-12). Following such an unsuccessful division, cells may continue to cycle or undergo cell-cycle arrest or death. This diversity of outcomes following treatment with antimitotic agents has been shown to be dependent on cell type as well as on concentration of the antimitotic agent used (Gascoigne and Taylor (2008) Cancer Cell 14:111-122; Orth et al. (2008) Mol Cancer Ther 7:3480-3489; Shi et al. (2008) Cancer Res 68:3269-3276).
The prolonged mitotic arrest model suggests that sustained high concentrations of drug are required for antitumor effect. Findings with weekly taxane therapies, which have equivalent efficacy to the once every three weeks taxane therapy schedule, suggest that the same effect can be obtained by splitting the total dose of drug administered.
The toxicities associated with paclitaxel and docetaxel are similar, and include neutropenia as the major dose limiting toxicity, along with significant peripheral neuropathy. In fact, dose reductions are frequent in heavily pretreated patients to mitigate the severity of these toxicities. In clinical studies dose reductions did not reduce the clinical response of the agents, suggesting that the optimal biological dose may be lower than the maximum tolerated dose (Salminen et al., (1999) J Clin Oncol 17:1127). Weekly administration of the taxanes has become more frequently used as clinical data demonstrated less myelosuppression with no decrease in clinical response (Gonzalez-Angulo et al., (2008) J Clin Oncol 26:1585). In breast cancer studies, weekly paclitaxel showed better response rates than once every three weeks dosing (Seidman et al., J Clin Oncol 26:1642 (2008)). However, weekly paclitaxel has demonstrated greater neuropathy than the once every three weeks schedule.
The cell division cycle also involves various protein kinases that are frequently overexpressed in cancer cells. Aurora A kinase, for example, is a key mitotic regulator that is implicated in the pathogenesis of several tumor types. The Aurora kinases, first identified in yeast (Ip11), Xenopus (Eg2) and Drosophila (Aurora), are critical regulators of mitosis. (Embo J (1998) 17, 5627-5637; Genetics(1993) 135, 677-691; Cell (1995) 81, 95-105; J Cell Sci (1998) 111 (Pt 5), 557-572). In humans, three isoforms of Aurora kinase exist, including Aurora A, Aurora B and Aurora C. Aurora A and Aurora B play critical roles in the normal progression of cells through mitosis, whereas Aurora C activity is largely restricted to meiotic cells. Aurora A and Aurora B are structurally closely related. Their catalytic domains lie in the C-terminus, where they differ in only a few amino acids. Greater diversity exists in their non-catalytic N-terminal domains. It is the sequence diversity in this region of Aurora A and Aurora B that dictates their interactions with distinct protein partners, allowing these kinases to have unique subcellular localizations and functions within mitotic cells.
Although Aurora B kinase and Aurora A kinase are both members of the Aurora kinase family, they have distinct roles during the process of mitotic division. In the course of normal mitotic cell division, cells organize bipolar spindles, with two radial arrays of microtubules each focused into a spindle pole at one end, and connected to chromosomes at the other end. In the instant before sister chromatids segregate into daughter cells, the chromosomes are arranged in a straight line (the ‘metaphase plate’). This process of organizing bipolar mitotic spindles with fully aligned chromosomes serves to ensure the integrity of a cell's chromosomal complement during mitosis.
The Aurora A gene (AURKA) localizes to chromosome 20q13.2 which is commonly amplified or overexpressed at a high incidence in a diverse array of tumor types. (Embo J(1998) 17, 3052-3065; Int J Cancer (2006) 118, 357-363; J Cell Biol (2003) 161, 267-280; Mol Cancer Ther (2007) 6, 1851-1857; J Natl Cancer Inst (2002) 94, 1320-1329). Increased Aurora A gene expression has been correlated to the etiology of cancer and to a worsened prognosis. (Int J Oncol (2004) 25, 1631-1639; Cancer Res (2007) 67, 10436-10444; Clin Cancer Res (2004) 10, 2065-2071; Clin Cancer Res (2007) 13, 4098-4104; Int J Cancer (2001) 92, 370-373; Br J Cancer (2001) 84, 824-831; J Natl Cancer Inst (2002) 94, 1320-1329). This concept has been supported in experimental models, demonstrating that Aurora A overexpression leads to oncogenic transformation. (Cancer Res (2002) 62, 4115-4122; Mol Cancer Res (2009) 7, 678-688; Oncogene (2006) 25, 7148-7158; Cell Res (2006) 16, 356-366; Oncogene (2008) 27, 4305-4314; Nat Genet (1998) 20, 189-193). Overexpression of Aurora A kinase is suspected to result in a stoichiometric imbalance between Aurora A and its regulatory partners, leading to chromosomal instability and subsequent transforming events. The potential oncogenic role of Aurora A has led to considerable interest in targeting this kinase for the treatment of cancer.
As a key regulator of mitosis, Aurora A plays an essential role in mitotic entry and normal progression of cells through mitosis. (Nat Rev Mol Cell Biol (2003) 4, 842-854; Curr Top Dev Biol (2000) 49, 331-42; Nat Rev Mol Cell Biol (2001) 2(1), 21-32). During a normal cell cycle, Aurora A kinase is first expressed in the G2 stage where it localizes to centrosomes and functions in centrosome maturation and separation as well as in the entry of cells into mitosis. In mitotic cells Aurora A kinase predominantly localizes to centrosomes and the proximal portion of incipient mitotic spindles. There it interacts with and phosphorylates a diverse set of proteins that collectively function in the formation of mitotic spindle poles and spindles, the attachment of spindles to sister chromatid at the kinetochores, the subsequent alignment and separation of chromosome, the spindle assembly checkpoint and cytokinesis. (J Cell Sci (2007) 120, 2987-2996; Trends Cell Biol (1999) 9, 454-459; Nat Rev Mol Cell Biol (2003) 4, 842-854; Trends Cell Biol (2005) 15, 241-250).
Although selective inhibition of Aurora A kinase results in a delayed mitotic entry (The Journal of biological chemistry (2003) 278, 51786-51795), cells commonly enter mitosis despite having inactive Aurora A kinase. Cells in which Aurora A kinase has been selectively inhibited demonstrate a variety of mitotic defects including abnormal mitotic spindles (monopolar or multipolar spindles) and defects in the process of chromosome alignment. With time, monopolar and multipolar spindles may resolve to form two opposing spindle poles, although some of these defects may lead immediately to cell death via defective mitoses. While spindle defects resulting from Aurora A kinase inhibition induce mitotic delays, presumably through activation of the spindle assembly checkpoint, cells ultimately divide at a frequency near that of untreated cells. (Mol Cell Biol (2007) 27(12), 4513-25; Cell Cycle (2008) 7(17), 2691-704; Mol Cancer Ther (2009) 8(7), 2046-56.). This inappropriate cell division occurs following a slow-acting suppression of the spindle assembly checkpoint due to loss of Aurora A kinase function. (Cell Cycle (2009) 8(6), 876-88). Bipolar spindles that are formed in the absence of Aurora A kinase function frequently show chromosome alignment and segregation defects, including chromosome congression defects at metaphase, lagging chromosomes at anaphase, and telophase bridges.
Consistent with the chromosome segregation defects, cells treated with MLN8054, a selective inhibitor of Aurora A kinase, develop aneuploidy that increases over time. Subsequent to repeated passages through defective mitotic divisions, cells treated with MLN8054 will often undergo senescence, an irreversible growth arrest with distinctive morphological characteristics. (Mol Cancer Res (2010) 8(3), 373-84). In some cell lines, MLN8054-treated cells exit from mitosis and activate a p53-dependent postmitotic G1 checkpoint, which subsequently induces p21 and Bax, leading to G1 arrest followed by the induction of apoptosis. (Mol. Cancer Ther (2009) 8(7), 2046-56). Some cells may also exit mitosis without cytokinesis. These cells enter the G1 phase of the cell cycle with double the normal DNA content and are therefore referred to as G1 tetraploid cells. Lastly, some cells may divide, albeit with severe chromosome segregation defects (Mol Cell Biol (2007) 27(12), 4513-25). In the latter two outcomes, the abnormal mitotic divisions result in deleterious aneuploidy leading to cell death or arrest. Alternatively, it is possible that a portion of these cells may be resistant to these terminal outcomes and can reenter the cell cycle, as aneuploidy has been demonstrated to be both a suppressor and a promoter of tumor cell growth.
Given the importance of the protein kinases involved in driving the cell cycle, it would be beneficial if more effective treatment regimens, which target these kinases could be developed. In particular, combined treatment regimens with antimitotics could be helpful for patients suffering from cell proliferative disorders, and might potentially even decrease the rate of relapse or overcome the resistance to a particular anticancer agent sometimes seen in these patients.
Drug tolerability and the prevalence of side effects are important considerations in structuring dose and schedule selection for the treatment of cell proliferative disorders. For example, treatments that require the use of therapeutic agents, for example, taxanes, that result in severe adverse events, such as neutropenia, may become ineffective due to insufficient patient compliance or because an effective therapeutic dose cannot be administered to the patient. Similarly, treatments that result in a higher effective concentration of the active ingredient for a longer period of time may provide increased therapeutic efficacy. Thus, there is a need for new cancer treatment regimens, including combination therapies, which avoid or ameliorate harsh side effects resulting from toxicity while providing increased therapeutic efficacy by achieving improved exposure efficacy.